Covalent attachment of polysaccharides that are potentially capable of eliciting protective antibodies, to carrier proteins to form optimum conjugate vaccines, is dependent upon both the size and structure of the two components. Methods for conjugating capsular polysaccharides, including the Vi capsular polysaccharide and pneumococcus types 6B, 12F, 19F, and 23F were devised using principles previously established in this and other laboratories as well as new joining reagents. The methods for conjugating polysaccharides of different chemical characteristics were developed. Polysaccharides having carboxyl group, single hydroxyl or vicinal hydroxyl groups were treated with different cross-linking reagents prior to their conjugation with carrier proteins. Dextran was used as model polysaccharide for study. Bacterial polysaccharide tested were: Vi, pneumococcus types 6B, 12F, 19F, 19A, and 23F, O-Specific side-chains of Shigella dysentariae type 1, Salmonella Paratyphi A, Staphylococcus aureus types 5 and 8. The Vi was modified by O-deacetylation and decarboxylation in order to study the relationship between structure and immunogenic epitopes. The immunogenicity of Vi conjugates, prepared with polysaccharides of large and low molecular weight and with the beta subunit and holotoxin of cholera toxin were compared in laboratory animals. In addition, clinical studies of a Vi-tetanus toxoid conjugate in adults were started.